I am looking to get some help on setting up a pipeline to analyze fluorescent images of transverse muscle sections. I am new to Cell Profiler, and additionally I believe my images present some unique challenges, so I’m looking to “poll the audience” to see what the best way to go about this endeavor is. I’ve attached a JPEG image that represents the kind of image I am trying to quantify (the TIF file was too large, so this pic is a low-res example) So here are my questions:
The picture you see is actually compiled from multiple photos taken at 10x. I used photoshop to stack the pictures one on top of the other (aligned the layers, then flattened the image, filled in the background). I’ve read elsewhere that using photoshop is a bad idea when you want to process the image on Cell Profiler, but I’m hoping that since I didn’t manipulate any of the colors (no manipulation of contrast, saturation, hue, etc.) that simply stacking the images will not affect the program’s functionality. I can always analyze individual pictures, however, so this problem could be avoided.
There are a couple of things I’ve stained for on this image. The first is laminin, which stains the outline of each individual muscle fiber. The blue stain is DAPI to identify all nuclei. The red is Pax7, a stain used to identify satellite cells (the stem cells of the muscle). Using these stains, i’m hoping I can quantify a number of things enumerated below:
A)The number of satellite cells in the image (colocalization of DAPI in blue, and Pax7 in red)
B)The total number of fibers in a transverse section
C)The total cross-sectional area of all of the muscle fibers.
D)The average cross-sectional area of an individual fiber.
E)The number of injured fibers (identified by centrally nucleated fibers [fibers with a DAPI stained nucleus located near the center of the fiber])
An additional consideration is that some areas of the transverse sections are not entirely perfect. I think I read somewhere that there is a way to manually exclude certain areas of the picture for analyzing. This would probably be a good idea in my case.
So, any suggestions out there to begin with? Also which one of the examples do you think would be best to model my pipeline after?
I’ll be reading through the manual and eagerly awaiting any suggestions. Thanks!